Isotypic neutralizing antibodies against mouse GCP-2/CXCL6 inhibit melanoma growth and metastasis
Introduction
Chemotactic cytokines, designated chemokines, are small secreted proteins which have the ability to attract leukocytes during physiological (e.g. lymphocytic homing) and pathological (e.g. inflammation and cancer) conditions [1], [2]. Chemokines contain conserved cysteine residues in their amino-terminal region, a characteristic used for classification into CXC, CC, CX3C and C chemokines [1], [2]. CXC chemokines are further subdivided in two groups depending on the presence or absence of an ELR (Glu-Leu-Arg)-motif located in front of the first conserved cysteine residue. Chemokines exert their biological functions through binding to G-protein coupled seven-transmembrane receptors designated as CXCR, CCR, CX3CR and CR [1], [3].
Human granulocyte chemotactic protein (huGCP-2)/CXCL6 was originally isolated from human MG-63 osteosarcoma cells by Proost et al. [4]. It is an ELR+ CXC chemokine which has been described to possess neutrophil chemotactic and angiogenic properties, similar to interleukin-8 (IL-8)/CXCL8, growth regulated oncogene (Gro)-α, β, γ/CXCL1, 2, 3 and epithelial neutrophil-activating peptide-78 (ENA-78)/CXCL5 [5], [6], [7]. In contrast, most CXC chemokines lacking the ELR-motif are angiostatic and chemoattract lymphocytes [5]. Human GCP-2 has 31% and 77% identical amino acids shared with IL-8 and ENA-78, respectively. Moreover, four different isoforms of huGCP-2 have been isolated from osteosarcoma cells: the mature 75 amino acid protein and three truncated forms missing two, five and eight NH2-terminal amino acids [4]. All four isoforms possess identical biological activities on granulocytes. However, compared with IL-8 they exhibit lower potency, but similar efficacy to attract granulocytes and to stimulate secretion of proteases such as matrix-metalloproteinase-9 (MMP-9) from their granules [4], [7]. In addition to MG-63 osteosarcoma cells, many other cancer types, including lung, prostate, breast and gastrointestinal carcinoma cells secrete huGCP-2 [8], [9], [10], [11]. Detection of increased amounts of huGCP-2 in cancer suggests that this chemokine may play a role during tumor development. This has been demonstrated by Van Coillie et al. by over-expression of murine GCP-2 (muGCP-2)/CXCL6 in human tumor allografts. This resulted in increased tumor growth due to enhanced angiogenesis and neutrophil chemoattraction [12]. HuGCP-2 has also been found to be produced by normal, non-transformed cells such as endothelial cells, fibroblasts and epithelial cells [13], [14], [15], [16], [17].
CXCR1 and CXCR2, to which huGCP-2 and IL-8 bind, mediate the neutrophil chemotactic and angiogenic activities [18], [19], [20]. Additionally, the affinity of huGCP-2 for CXCR2 is higher than that for CXCR1, suggesting that activation of CXCR2 initiates neutrophil migration distant from the site of inflammation, whereas CXCR1 is involved in the inflammation area where high concentrations of huGCP-2 are present [7].
Up to now, IL-8 has not yet been identified in mice. However, muGCP-2 isolated from mouse fibroblasts and epithelial cells, has been identified as one of the most potent neutrophil attracting chemokines in mice [7], [21], [22], [23]. MuGCP-2 has more than 60% amino acid identity with huGCP-2 and it is more active than huGCP-2 to attract human neutrophils. In addition, muGCP-2 is an equally potent chemoattractant as IL-8 on mouse neutrophils [7]. This suggests that muGCP-2 is functionally the murine counterpart for human IL-8. Furthermore, muGCP-2 can be processed both at its NH2- and COOH-terminus and up to 28 different isoforms have been isolated [23]. In addition, it has been described that NH2-terminal truncations and COOH-terminal cleavage of muGCP-2 increases the chemotactic activity [23]. As a consequence, muGCP-2(9-78) has been established to be the most active and the muGCP-2 precursor LIX (lipopolysaccharide-induced CXC chemokine) the least active neutrophil chemoattractant [23]. Furthermore, muGCP-2 exerts its functions through binding to muCXCR1 and muCXCR2, whereas murine Gro-α/keratinocyte chemoattractant (KC)/CXCL1 and murine Gro-β,γ/macrophage inflammatory protein (MIP)-2/CXCL2,3, two other neutrophil chemoattractants, bind only mCXCR2 [24], [25], [26]. This is in line with the thesis that muGCP-2 is the functional mouse homologue of huIL-8.
One way to decipher further the role of GCP-2 in tumor biology is by knocking out its functions. The purpose of this study was to neutralize the neutrophil chemotactic and angiogenic activities of GCP-2 in vivo. Specific mouse monoclonal antibodies against murine GCP-2 were therefore derived from C57Bl/6 mice immunized with muGCP-2(9-78) cross-linked to ovalbumin. After isolation of spleen cells, hybridoma cell cultures were created from which the specific monoclonal anti-muGCP-2 antibodies were isolated. This technique has previously been used to generate mouse monoclonal antibodies against muIL-17 and muIL-12 [27], [28]. To determine the neutralizing capacity and specificity of these antibodies in vitro, chemotaxis and calcium assays were performed. To demonstrate that the anti-muGCP-2 antibodies were not antigenic and remained active in the circulation pharmacokinetic studies were performed. Furthermore, we were able to demonstrate that anti-muGCP-2 antibodies inhibited tumor growth and metastasis in vivo.
Section snippets
Reagents
Murine GCP-2(9-78) which lacks the first eight amino acids was prepared in Escherichia coli cells and purified by a three-step purification procedure as described before [12]. KC and MIP-2 were purchased from Peprotech (Rocky Hill, NY). Monoclonal mouse anti-muGCP-2 antibodies, mGCP-2 Ab4 (mAb4) an IgG2b and mGCP-2 Ab5 (mAb5) an IgG2a, and isotype mouse IgG2b antibodies were developed and purified as previously described for anti-IL-17 [27]. Polyclonal rabbit anti-muGCP-2 antibodies (Rega
Specific neutralization of muGCP-2 activity in vitro
To investigate the neutralizing capacity of the murine monoclonal antibodies which were generated to block muGCP-2(9-78) activity, receptor signaling and chemotaxis (Boyden) assays on neutrophilic granulocytes were performed. For such inhibition experiments, muGCP-2(9-78) protein was pre-incubated with monoclonal or polyclonal antibodies before addition to the test system. Fig. 1A shows that granulocyte migration toward muGCP-2(9-78) is inhibited after addition of monoclonal anti-GCP-2
Discussion
The cellular composition of tumors is complex, and comprises tumor cells, fibroblasts, endothelial cells as well as tumor infiltrated leukocytes. Tumor-associated macrophages and neutrophils may favor tumor progression and metastases by secretion of growth factors and matrix-degrading enzymes, respectively [32], [33]. Alternatively, infiltration of dendritic cells, cytotoxic T lymphocytes and natural killer (NK) cells is rather detrimental for tumor development [34], [35]. Endothelial cells
Conflict of interest
None declared.
Acknowledgements
This work was supported by the Centers of Excellence (Credit No. EF/05/15) of the K.U.Leuven, the Concerted Research Actions (G.O.A.) of the Regional Government of Flanders, the Fund for Scientific Research of Flanders (F.W.O. Vlaanderen), the Fonds National de la Recherche Scientifique Médicale (FRSM, Belgique), the European Union 6FP EC contract INNOCHEM and the Interuniversity Attraction Poles Program (I.A.P.)-Belgian Science Policy. The authors thank Jean-Pierre Lenaerts, René Conings,
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